Platelet lysate gel

ABSTRACT

The invention concerns a pharmaceutical composition comprising a platelet lysate and its use to treat a wound, an anal fissure, vaginal atrophy or a wrinkle.

FIELD OF THE INVENTION

The invention concerns a pharmaceutical composition comprising aplatelet lysate and its use to treat a wound, an anal fissure, vaginalatrophy or a wrinkle.

BACKGROUND OF THE INVENTION

Approximately 15% diabetics will have an ulcer in their lifetime.Annual, population-based incidence of 1% to 3.6% among people with type1 or type 2 diabetes. By 2030, approximately 13 million people willsuffer a diabetic foot ulcer each year. Foot ulcers precede more than84% of non-traumatic lower limb amputations. In addition, the 3-yearmortality after a first amputation has been estimated as high as 20-50%.The average cost of healing a single ulcer is $8,000, that of aninfected ulcer is $17,000, and that of a major amputation is $45,000.The excess cost attributed to foot ulcers and their sequelae averaged$27,987 per patient for a 2-year period following ulcer presentation(American Diabetes Association; US Department of Health and HumanServices; Diabetes Care; Journal of Clinical investigations; JAMA293:217-228, 2005; Diabetes Care 22:157-162, 1999).

Free blood flow to a site of injury provides access for signalingmolecules and nutrients. Regulation of proliferation and remodelling ofcollagen formation requires coordination of molecular processes (e.g.protease and protease inhibitor balance). Immune and inflammatoryresponse ensure bacteria and debris are phagocytosed and removed preventinfection (J Invest Dermatol. 2007 May; 127(5): 1018-29). If untreated,a diabetic ulcer can progress from a small irritated but unbroken skinpatch to a potentially life-threatening wound involving extensive tissuedeath and infection. Treatment of the diabetic skin ulcer may includedrying out the wound, debriding (excising) the dead tissue, andadministering systemic antibiotics. (American College of Foot and AnkleSurgeons; Medscape website; Clinical Diabetes Spring 2009 vol. 27 no. 252-58).

In regard to the use of platelet lysate in cosmetics it is well knownthat healthy skin requires tight coordination at the physiological,cellular and molecular levels. Three critical functions required forsustained healthy skin include functioning connective tissue cells,intact skin extracellular matrix (ECM), and tight molecular regulationof the proliferation and remodeling process.

The process to form new tissue requires the action and balance of anumber of factors including proteases, tissue inhibitors, and pro- andanti-inflammatory molecules. Coordinated regulation of these factorsensures permanent collagen tissue is being generated while the temporaryscaffold is degraded.

Functioning Connective Tissue Cells

Cells within the skin, such as fibroblasts and keratinocytes, arerequired to synthesize, maintain and provide the extracellular matrix(ECM) that functions as the structural framework of the skin. It isespecially important for fibroblasts, which produce the majority ofcollagen in the skin, to remain healthy and elastic [1]. Platelet andgrowth factor based solutions enhance wound healing by increasing localconcentration of beneficial factors [2, 3]. Current therapies areinvasive or minimally accelerate the process of wound healing. Anadvanced dressing such as hydrocolloid gel have limited improvement overtraditional non-adherent gauze in the treatment of diabetic ulcers [6].Technological breakthroughs in wound healing have also been limited overthe last 15 years, with little improvement regarding patientquality-adjusted life year (QALY) outcomes [7].

In healthy skin, intact type I collagen fibrils in the dermis providemechanical stability and attachment sites for fibroblasts. Receptors(integrins) on the surface of fibroblasts attach to collagen (and otherproteins in the dermal extracellular matrix). Cytoskeletal machinery(actin-myosin microfilaments, not shown) within fibroblasts pulls on theintact collagen matrix, which in turn offers mechanical resistance.Dynamic mechanical tension that is created promotes assembly ofintracellular scaffolding (microtubules/intermediate filaments, notshown), which pushes outward to cause fibroblasts to stretch. Thisstretch is required for fibroblasts to produce normal levels of collagenand proteases.

Intact Skin Extracellular Matrix (ECM)

A healthy balance of intact collagen (the most abundant protein in ECM),elastin, hyaluronic acid, proteoglycans, fibronectin, and laminin arenecessary for healthy, wrinkle-free skin. These components create ascaffold to support the skin and give it an evenly distributed texture[8].

Tight Molecular Regulation of the Proliferation and Remodelling Process.Forming new tissue requires the action and balance of a number offactors including proteases, tissue inhibitors, and pro- andanti-inflammatory molecules. Coordinated regulation of these factorsensures permanent collagen tissue is being generated; damaged moleculesare cleaved and cleared, and overall health of the connective tissue[9].

All fibrillar collagens consist of three polypeptide chains wound aroundeach other in a triple helical configuration. The soluble triple helix,which is termed procollagen, is assembled inside fibroblasts.Procollagen is secreted from fibroblasts, and the peptide ends areremoved by two enzymes in the extracellular space [10]. Removal of theends produces collagen, which spontaneously assembles (i.e., matures)into large fibres that are enzymatically cross-linked. Thiscross-linking is necessary for normal structural support [11]. Type Icollagen undergoes natural breakdown by enzymatic degradation; however,this degradation in human skin is exceedingly slow [12]. Humans expressonly four enzymes that are capable of initiating breakdown of type Icollagen [13].

These collagenases are members of a family of matrix protein-degradingenzymes, referred to as matrix metalloproteinases (MMPs) [14]. MMPs areresponsible for physiological degradation of various extracellularmatrix proteins [12]. Of the four collagenases that are expressed inhumans, only interstitial collagenase (MMP-1) is involved in normalturnover of skin collagen [14]. In healthy young skin, MMP-1 expressionis exceedingly low, near the limit of detection by the most sensitivemeasurement methods.

Once cleaved by MMP-1, collagen unravels, then un-raveled collagen,called gelatin, then undergoes further degradation by other members ofthe MMP family, called gelatinases. These gelatinases are also expressedat very low levels in normal skin [14, 15]. In addition, skin expressesnatural inhibitors of these MMPs. These tissue inhibitors of matrixmetalloproteinases (TIMPs) further act to retard collagen breakdown.Thus, type I collagen in human skin is very stable, requiringapproximately 30 years on average to undergo replacement [1, 12].

Wrinkles

Wrinkles are caused by habitual facial expressions, aging, sun damage,smoking, poor hydration and various other factors. Under stress,fibroblasts produce less ECM molecules and more molecules which breakdown the existing matrix. This leads to further malfunctioning of theconnective tissue cells in a feedback loop.

Malfunctioning of Connective Tissue Cells

A delicate relationship exists between mechanical tension, collagensynthesis and collagen fragmentation by collagenase (COLase) in humanskin. In aged human skin, attachments of fibroblasts to integrins arelost and fragmented collagen fibrils fail to provide sufficientmechanical stability to maintain normal mechanical tension. Reducedmechanical tension causes fibroblasts to collapse, and collapsedfibroblasts produce less procollagen and more collagenase (COLase).Reduced collagen production and increased collagenase-catalysed collagenfragmentation result in further reduction of mechanical tension, therebycausing continual loss of collagen [1].

Breakdown of the Existing ECM

The existing ECM molecules, especially collagen fibres, are broken downthrough physical, chemical, or proteolytic damage, leaving collagen andother molecular fragments throughout the ECM.

The slow rate of type I collagen turnover allows accumulation ofage-dependent modifications that impair its functions. These alterationsinclude formation of new cross-links derived from sugars [14].Importantly, these crosslinks are not able to be efficiently broken downand removed during the slow normal process of MMP-mediated turnover,causing accumulation of fragmented collagen within the extracellularmatrix as skin ages [15, 16]. Cross-links prevent complete removal ofcollagen fragments. The fragments cannot be repaired or incorporatedinto newly made collagen fibrils, and therefore cause defects in thethree dimensional collagen matrixes. These defects impair the structuraland mechanical integrity of the dermis and thereby deleteriously alterits function. Accumulation of fragmented collagen lies at the heart ofage-related changes in the appearance of human skin

Deregulated Molecular Signaling

Collapsed fibroblasts and degraded ECM disproportionately increase theconcentration of matrix metalloproteases to tissue inhibitors ofmetalloproteases (TIMP) at the wrinkle site, impeding tissueregeneration [1, 10-20].

Reactive oxygen species (ROS), a by-product of both environmentallyinduced and intrinsic aging, cause a cascade of biochemical reactionswithin the skin, which results in the production of matrixmetalloproteinases (MMPs) and proinflammatory cytokines. MMPs, secretedby fibroblasts and keratinocytes, decrease collagen formation andenhance collagen degradation, contributing to the breakdown of thedermal matrix [19].

Current Wrinkle Therapies

Current treatments on the market fail, they do not recreate youthfulappearance with lasting results. They aim to treat the symptoms ofwrinkles without addressing the root causes. Toxins temporarily relaxmuscles in the face that stretch the skin, but does not repair theunderlying extracellular matrix. Botox® and Dysport® can result in‘frozen face,’ with problems swallowing, speaking, and breathing.Fillers (such as hyaluronic acid, collagen, poly-L-lactic acid, andhydroxylapatite) ‘fill the gaps’ for a short period, but does notrestore skin to normal physiology or prevent the degenerative cycle.

Technological breakthroughs in cosmetics have been limited over the last30 years, with sporadic advancements over the last decade. First andsecond generation injectable therapies have a quick time to onset, but alimited duration of effect.

Platelet Lysate Therapies

There are several long standing issues affecting the wide spreadadaptation and use of platelet lysate (PL) in the clinic. The mainissues have been, but are not limited to the following: donor derivedsamples run the risk of contamination during processing; plateletquality varies from patient to patient in terms of count and ability tosecrete beneficial growth factors making the preparations inconsistent;there is a need for platelet quantitation in physician office kits;therapeutic preparation introduces high patient-by-patient variabilityaffecting efficacy due to intrinsic differences in platelet count;current methods are inconvenient, clinicians must centrifuge blood toisolate platelets from blood, and the variability of this process, whichcan last between 25-30 minutes, again introduces errors; and the currentprocesses are not cost-effective with an estimated cost of $130 pertreatment (Plateltex). The invention is a non-obvious solution to theselong standing and persistent problems.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a flowchart of the protocol used to produce a plateletlystate of the invention and a pharmaceutical composition of theinvention.

FIGS. 2 and 3 show that the invention provides a cost-effective,differentiated product that is convenient, consistent and cGMP compliant

FIGS. 4 to 7 show that the solves the long standing and presistent needto provide a consistent wound care product that is quality controlled,convenient to use and cost effective.

SUMMARY OF THE INVENTION

The invention provides a pharmaceutical composition comprising aplatelet lysate. These may be therapeutically applied to wounds, such asulcerated wounds, anal fissures, vaginal atrophy or wrinkles.

The inventors have also devised a more efficient way of lysing plateletssuch they release their growth factors into solution as a plateletlysate. The novel platelet lysate may itself be used as a therapeutic ormay be formulated into a pharmaceutical composition of the invention.

The composition or lysate of the invention may be lyophilised to producea stabilized, freeze-dried powder that contains these growth factors andwhich can be used for therapeutic application in wound healing, but alsoother therapeutic applications in which there is an increased need fordelivery of natural growth factors.

The invention provides a pharmaceutical composition comprising (a) atherapeutic platelet lysate, (b) at least one pharmaceuticallyacceptable polymer and (c) at least one pharmaceutically acceptablepositively charged chemical species selected from the group consistingof lysine, arginine, histidine, aspartic acid, glutamic acid, alanine,methionine, proline, serine, asparagine, cysteine, polyamino acids,protamine, aminoguanidine, zinc ions and magnesium ions, wherein thecomposition is an aqueous gel having a viscosity in the range of 1000 to500,000 mPa·s (cps) at room temperature.

The invention also provides:

-   -   a method of producing a platelet lysate comprising subjecting a        population of platelets to at least one freeze-thaw cycle,        wherein the freeze portion of each cycle is carried out at a        temperature lower than or equal to −78° C.;    -   a method of producing platelet lysate comprising subjecting a        population of platelets to mechanical homogenisation (‘waring        blender’), liquid homogenization, sonication, freeze-thaw using        alcohol and dry ice or other means, mechanical grinding in        liquid nitrogen at >75%, but typically >90%, lysis to release        growth factors from platelets and alpha granules into solution        whereas rotating blades grind and disperse cells and tissues;        cell or tissue suspensions are sheared by forcing them through a        narrow space; high frequency sound waves shear cells; repeated        cycles of freezing and thawing disrupt cells through ice crystal        formation; grinding tissue, frozen in liquid nitrogen wherein        lysis releases growth factors from platelets and alpha granules        into solution;    -   a platelet lysate produced using a method of the invention;    -   a pharmaceutical composition of the invention or a platelet        lysate of the invention for use in treating a wound, an anal        fissure, vaginal atrophy or a wrinkle in a patient in need        thereof;    -   a method of treating a wound, an anal fissure, vaginal atrophy        or a wrinkle in a patient in need thereof, comprising        administering to the patient a therapeutically effective amount        of a pharmaceutical composition of the invention or a platelet        lysate of the invention;    -   a method of producing a pharmaceutical composition of the        invention comprising mixing a platelet lysate with at least one        pharmaceutically acceptable polymer and at least one        pharmaceutically acceptable positively charged chemical species        such that the resulting composition is an aqueous gel having a        viscosity in the range of 1000 to 500,000 mPa·s (cps) at room        temperature; and    -   a method of producing a pharmaceutical composition of the        invention comprising (a) producing a platelet lysate using a        method according to any one of claims 14 to 20 and (b) mixing        the platelet lysate with at least one pharmaceutically        acceptable polymer and at least one pharmaceutically acceptable        positively charged chemical species such that the resulting        composition is an aqueous gel having a viscosity in the range of        1000 to 500,000 mPa·s (cps) at room temperature.

DETAILED DESCRIPTION OF THE INVENTION Invention Details

The pharmaceutical composition of the invention or the platelet lysateof the invention will be applied, and all components will undergorigorous testing to ensure that our product is safe and effective forthe patient. These processes include platelet screening and methods toallow for the production of sterile platelet gel. The inventionimplements processes which are safe, effective, and sterile. In apreferred embodiment, sterile platelet lysate is mixed with sterilemethylcellulose gel to create a therapeutic platelet composition of theinvention. A flowchart of the protocol of the invention can be found inFIG. 10.

Effectiveness of Pharmaceutical Composition of the Invention forWrinkles

The invention provides a regenerative therapy that directly addresseseach of the problems associated with wrinkles and enhances the skin andthe underlying scaffold. Treatment with a pharmaceutical composition ofthe invention or a platelet lysate of the invention reverses damagedskin's degenerative cycle to the healthy physiology found in normalskin. The pharmaceutical composition of the invention or platelet lysateof the invention works by rebalancing cells within the connectivetissues, equilibrating molecular signalling, and restoring extracellularmatrix. The natural healing and tissue regeneration process leads toincreased collagen synthesis, regeneration of the collagen extracellularmatrix and proliferation of the fibroblasts within the matrix.

Rebalanced Cells within the Connective Tissues

Since the pharmaceutical composition of the invention is derived fromplatelets, it typically comprises various growth factors which arederived from platelets and promote cell growth. The pharmaceuticalcomposition of the invention or the platelet lysate of the inventiontypically comprises one or more of, preferably all of, the followinggrowth factors: PDGF, VEGF, FGF, EGF, TGF, especially TGF-β, and CTGF.The composition preferably comprises 2, 3, 4, 5 or 6 of these growthfactors.

Platelet-derived growth factor (PDGF) promotes cell growth andgeneration, repair of blood vessels and collagen production. Vascularendothelial growth factor (VEGF) promotes growth and generation ofvascular endothelial cells. Fibroblast growth factor (FGF) promotestissue repair, cell growth, collagen production and hyaluronic acidproduction. Epithelial growth factor (EGF) promotes epithelial cellgrowth, angiogenesis and wound healing. Transforming growth factor(TGF), especially TGF-β, promotes growth and neogenesis of epithelialcells and wound healing. Connective tissue growth factor (CTGF) promoteswound repair.

The pharmaceutical composition of the invention or the platelet lysateof the invention therefore promotes the formation of new fibroblasts.These new fibroblasts start elastic and healthy, producing new collagenand less metalloproteases. The restoration of fibroblasts (the majorcell in synthesizing, maintaining and providing the structuralframework) results in healthier, restored skin [1].

PDGF has also been show to increase fibroblast motility, allowingfibroblasts to relocate to the site of administration.

Restored Extracellular Matrix (ECM)

Natural growth factors found in the alpha-granules of platelets (such asPDGF, VEGF, FGF, EGF, and TGF) promote collagen and hyaluronic acidproduction, tissue repair, growth and regeneration of endothelial cellsand epithelial cells, and new blood vessel formation (which restoresoxygen and removes undesired molecules). All of these factors help toregenerate wrinkled and damaged ECM back to its healthy state. Each ofthese growth factors plays a role within skin regeneration andrestoration, both individually and additively in concert with eachother. Treatments that stimulate the production of new, non-fragmentedcollagen will provide substantial improvement to the appearance andhealth of aged [1].

Equilibrated Molecular Signalling

Application of platelet lysate under the wrinkle provides a concentrateddose of TIMP protein to address the metalloprotease imbalance, preventsthe further degradation of ECM, and increases the speed of skinregeneration. Direct application of platelet lysate to skin providesconcentrated dose of TIMP protein to address the metalloproteaseimbalance, supporting collagen tissue proliferation and remodelling.Platelets contain for example TIMP-2 which inhibits most MMPs, butpreferentially inhibits MMP-2 and MMP-9 [20]. The pharmaceuticalcomposition of the invention or the platelet lysate of the inventiontypically comprises TIMP metallopeptidase inhibitor 2 (TIMP-2).

Advantages of the Invention

The pharmaceutical composition of the invention or the platelet lysateof the invention will be easily adapted for the chronic wound healingmarkets and conveys the following advantages over platelet rich plasmaand other autologous regenerative cell therapies.

1. Production

In-house, scaled-up cGMP compliant in-vitro platelet production platformto deliver high yields of safe, pure and active platelets.

2. Formulation

Platelets are lysed to release important growth factors. Lysate isconcentrated to standard efficacious therapeutic dose and freeze-driedfor long term storage at refrigerated temperature. QC testing onartificial skin assay ensures consistent, high concentration of growthfactors in every batch and no inter-patient variability seen withautologous platelet rich plasma.

3. Administration

Efficient one-step processes of (a) administering a pharmaceuticalcomposition of the invention or a platelet lysate of the invention, (b)rehydrating a freeze-dried platelet lysate of the invention, (c) mixinga freeze-dried platelet lysate of the invention with polymer gellingfactor or (d) rehydrating a pharmaceutical composition of the inventionsimplifies current process for physicians in the clinic and standardizespatient dosing.

Processing for current platelet rich plasma therapy includes spinningblood and isolating un-concentrated, un-characterized plasma, whichvaries by patient on growth factor concentration. Unlike other dermalstimulators (e.g., poly-L-lactic acid), platelet therapy produces itseffect rapidly. It is reproducible and easy to use.

Additional advantages are shown in Table 1 below.

TABLE 1 Advantages of the therapeutic composition of the invention(labeled 4^(th) generation) vs. previous generation products Table showsmajor advantages to CTL's fourth generation platelet-rich plasma gelClinical Contamination Unwanted Long-term # of Growth Use withGeneration Product Example Price Convenience Risk Impurities PotencyStability Factors sickly² patient 1^(st) Manufactured Regranex ✓ ✓ ✓ ✓ x✓ x ✓ growth factor 2^(nd) Autologous (Self) Autologel x x x x  x¹ x  x¹x platelet gel 3^(rd) Allogenic (Donor) Academic x ✓ ✓ x ✓ x ✓ ✓platelet gel Trials 4^(th) Allogenic (Donor) CTL ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓platelet gel with methylcellulose ¹Variable by patient ²Abnormal bloodor platelet physiology

Platelet therapies are a natural, sustained solution, while knownproducts are unnatural and temporary, the pharmaceutical composition ofthe invention and platelet lysate of the invention provide a bettersolution to patients, with fewer visits and cost-savings. Unlike knownproducts, the pharmaceutical composition of the invention and plateletlysate of the invention standardize the concentration of platelets andgrowth factors, leading to consistent results.

Additionally, the therapeutic composition solves hurdles for providing aconsistent dermal regeneration product that is quality controlled,convenient to use and cost effective.

-   PRODUCTION: The composition of the invention is a cGMP compliant,    standardized product that will ensure a consistent dermal    regeneration outcome.-   USABILITY: The composition of the invention is a one-step    application platelet lysate formulation and will save significant    time for the physician.-   COST: The composition of the invention will deliver dermal    regeneration at a cost-effective price when compared with other    platelet-rich plasma therapies.

Other non-obvious solution to the problem to be solved are there are noother commercial platelet lysate/methylcellulose gels on the market andthere are no other platelet lysate/methylcellulose gels in theliterature.

Diabetic Ulcers

Diabetic patients often suffer from peripheral arterial disease thatconstricts blood flow, which restricts access of signaling molecules andnutrients to the wound site. High concentration of cytokines increasethe concentration of metalloproteases to inhibitors at the wound site,impeding normal tissue regeneration. Poor activation ofimmune-modulatory factors combined with low levels of bactericidalnitric oxide increases probability and severity of infections [21].While the exact process of wound healing is still not fully understood,it is known that platelets secrete a subset of factors critical to theprocess. Factors important for wound healing are naturally secreted byplatelets, macrophages and fibroblasts. PDGF, EGF and TGF are consideredcritical factors in the process of wound healing. Additional factorsplay important role and are secreted by macrophages and fibroblastsrecruited to wound site by platelets. Skin is presumably the organ mostsubject to injury. Skin repair is a complex process that can be dividedin 4 phases usually described as inflammation, granulation tissueformation, and epithelialization and remodelling of the connectivetissue matrix. Each of these phases is complex in itself, and it isclear that for good wound healing, the processes must occur successivelyand in coordination. Good wound healing can be defined as restoration ofthe skin, including the dermal and epidermal part, in such a way thatthe resulting scar tissue maximally resembles the unwounded skinstructurally, histologically, functionally, and aesthetically obviously,such scar tissue is different from a hypertrophic scar or keloid.

For purposes of clarity a simplified description of the composition ofhuman skin is given below. The upper part is composed of the epidermis,which contains mostly epithelial cells, some. Five different layers arefound in the epidermis. The base of the epidermis, i.e., in the stratumbasal, is attached to the dermis via the basement membrane. The dermisis composed of connective tissue, including fibroblasts and otherconnective tissue cells, and connective tissue matrix substances. Bloodvessels, nerves, sensory organs, sweat glands, sebaceous glands, andhair follicles are present in the dermis. Clinical experiments havedemonstrated that application of platelet lysate preparations induceswound healing in chronic wounds such as ulcers and in burns.

Diabetic ulcers are a result of neuropathy and decreased circulation inthe lower limbs of the diabetic patient. These difficult to heal woundsform a vast amount of exudate that currently is being managed byabsorbing the majority in specific wound dressings. Exudate contains alarge variety of proteins and cells. One of these proteins is thrombin(current hypothesis, needs to be confirmed) a component of thecoagulation cascade which activates platelets. Platelets are small cellsin the blood which form blood clots upon activation and release theirgrowth factor content. They are activated by tissue damage and the bloodclot prevents further bleeding. The growth factors promote regenerationand repair of the damaged tissue. The wound healing capacity ofplatelets has tried to be captured by many researchers in a wide varietyof formulations such as platelet-rich plasma, platelet lysate, plateletgel and platelet. These products are prepared by in vitro activatedplatelets which are then prepared into a suitable formulation andapplied to the wound.

The therapeutic platelet concentration has been described to be 1×10⁹platelets per ml, which we achieve by isolating and concentrating theplatelets through centrifugation. The ideal concentration needs to bedetermined through clinical evaluations, but is likely to range from theconcentration in platelet-rich plasma which is roughly the double ofthat in peripheral blood (i.e. 4×10⁸ platelets/ml if the patient's bloodlevel is 2×10⁸ platelets/ml), to 2×10⁹ platelets/ml. The Gold Standardhas been to store isolated platelets at room temperature whilst theyagitate until they are processed (standard guidelines followed at theWelsh Blood Service).

Platelet Lysate

Platelet lysate, PL, has been shown to have a positive influence onwound healing. A therapeutic platelet lysate is a platelet lysate thatis suitable for therapy. The platelet lysate of the invention or theplatelet lysate used in the pharmaceutical composition of the inventiontypically comprises one or more of, preferably all of, PDGF, VEGF, FGF,EGF, and TGF as described above. It also typically comprises TIMP-2.

Lysis of platelets can be accomplished through chemical means (i.e.CaCl2), osmotic means (use of distilled H2O), or throughfreezing/thawing procedures. Platelet lysate for use in the inventioncan also be derived from whole blood and can be prepared as described inU.S. Pat. No. 5,198,357 [22], which is incorporated by reference herein.

Previously, PL has been prepared through freeze-thaw cycles in varyingtemperatures ranging from −20 to −80 degrees, by rarely using more than1 cycle. Our data have shown that the number of whole platelets is onlymoderately affected by a freeze-thaw cycle in −80/+37 degrees. In fact,in a comparison of −20, −80 and liquid nitrogen freezing, only liquidnitrogen was able to achieve 90% lysis whereas −20 and −80 resulted in80% and 50% lysis, respectively. Notable however is that the experimentabove contained 3 freeze-thaw cycles and thus shows that the preparationof PL in current literature using −80 degrees in one freeze-thaw canonly achieve about 20% lysis. Liquid nitrogen also holds the advantageof enabling 3 or more freeze-thaw cycles to be performed within an hour,and is thus a highly suitable method for the clinical setting.Furthermore, liquid nitrogen facilitates process standardization as theliquid form of the gas always is −196° C. whereas freezers can differ intemperature over time, especially when there are multiple users who openthe door frequently naturally standardization is of particularimportance when preparing a product for use in the clinic.

The invention provides a method of producing a platelet lysate (PL)comprising subjecting a population of platelets to at least onefreeze-thaw cycle, wherein the freeze portion of each cycle is carriedout at a temperature lower than or equal to −78° C. The method mayinvolve any number of freeze-thaw cycles, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30 or more. The method preferably comprisessubjecting the population of platelets to 5 or fewer freeze-thaw cycles,such as 4 or fewer freeze-thaw cycles, 3 or fewer freeze-thaw cycles or2 or fewer freeze-thaw cycles. The method more preferably comprisessubjecting the population of platelets to only one freeze-thaw cycle,only two freeze-thaw cycles, only three freeze-thaw cycles or only fourfreeze-thaw cycles.

The thaw temperature in each cycle may be any temperature that thaws theplatelet composition, such from about 5° C. to about 50° C., such asfrom about 10° C. to about 45° C. or from about 20° C. to about 40° C.The thaw temperature in each cycle is typically about 37° C.

The freeze temperature in each cycle is preferably lower than or equalto about −79° C., lower than or equal to about −80° C., lower than orequal to about −81° C., lower than or equal to about −82° C., lower thanor equal to about −83° C., lower than or equal to about −84° C., lowerthan or equal to about −85° C., lower than or equal to about −86° C.,lower than or equal to about −87° C., lower than or equal to about −88°C., lower than or equal to about −89° C., lower than or equal to about−90° C., lower than or equal to about −100° C., lower than or equal toabout −110° C., lower than or equal to about −120° C., lower than orequal to about −130° C., lower than or equal to about −140° C., lowerthan or equal to about −150° C., lower than or equal to about −160° C.,lower than or equal to about −170° C., lower than or equal to about−180° C., lower than or equal to about −190° C. or about −196° C.

The freeze and thaw temperatures in different cycles of the same methodare typically the same.

In a preferred embodiment, liquid nitrogen is used as a cryogenic meansin each freeze cycle. Immersion in liquid nitrogen in the freeze portionof each cycle typically results in 95% or more lysis of plateletsresulting in greater growth factor release and improved function in cellgrowth, repair and regeneration measurable by, but not limited to,fibroblast and PGDF assay.

The invention also provides a platelet lysate produced using a method ofthe invention. This platelet lysate differs from known lysate because itresults from greater than about 80% lysis of the platelets, such asgreater than about 85% or greater than about 90% lysis of the platelets.It therefore comprises more growth factors and an improved function incell growth, repair and regeneration measurable by, but not limited to,fibroblast and PGDF assay.

The platelet lysate is present in the pharmaceutical composition of theinvention at a concentration within the range of from about 0.1 μg toabout 1,000 μg per gram of composition, such as from about 0.2 μg toabout 750 μg per gram of composition, from about 0.5 μg to about 500 μgper gram of composition, from about 1 μg to about 250 μg per gram ofcomposition, from about 2 μg to about 200 μg per gram of composition orfrom about 10 μg to about 100 μg per gram of composition.

The platelet lysate of the invention or the platelet lysate used in thepharmaceutical composition of the invention may be derived from theplatelets of any mammal. The mammal is preferably a human. However, itmay be non-human. Suitable non-human animals include, but are notlimited to, primates, such as marmosets or monkeys, commercially farmedanimals, such as horses, cows, sheeps, goats, alpacas, guanacos, deer orpigs, pets, such as dogs, cats, mice, rats, guinea pigs, ferrets,gerbils or hamsters or wild animals such as badgers or deer.

Pharmaceutically Acceptable Polymer

The pharmaceutical composition of the invention comprises at least onepharmaceutically acceptable polymer. The composition may comprise anynumber of pharmaceutically acceptable polymers, such as 1, 2, 3, 4, 5,10 or more.

A polymer is pharmaceutically acceptable if it suitable for use intherapy. The polymer is preferably suitable for topical administrationto a wound, such as any of the wounds discussed herein, an anal fissure,vaginal atrophy or a wrinkle.

Pharmaceutically acceptable polymers are well known in the art. Any suchpolymers may be used in accordance with the invention.

The polymer concentration is preferably from about 15% (w/w) to about30% (w/w), such as from about 17% (w/w) to about 25% (w/w) or from about20% (w/w) to about 23% (w/w).

The polymer is preferably a cellulose polymer. Suitable cellulosepolymers are know in the art. The cellulose polymer iscarboxymethylcellulose, hydroxypropylmethylcellulose or methylcellulose.The cellulose polymer concentration is preferably from about 1.5% (w/w)to about 4.0% (w/w), such as from about 2.0% (w/w) to about 3.0% (w/w).The cellulose polymer preferably has a molecular weight of from about450,000 to about 4,000,000, such as from about 500,000 to about3,500,000, from about 500,000 to about 3,000,000 or from about 750,000to about 2,500,000 or from about 1000,000 to about 2,000,000.

The polymer is preferably a pluronic acid, optionally Pluronic F-127.

Pharmaceutically Acceptable Positively Charged Chemical Species

The pharmacetucal composition of the invention comprises at least onepharmaceutically acceptable positively charged chemical species selectedfrom the group consisting of lysine, arginine, histidine, aspartic acid,glutamic acid, alanine, methionine, proline, serine, asparagine,cysteine, polyamino acids, protamine, aminoguanidine, zinc ions andmagnesium ions.

A species is pharmaceutically acceptable if it suitable for use intherapy. The species is preferably suitable for topical administrationto a wound, such as any of the wounds discussed herein, an anal fissure,vaginal atrophy or a wrinkle.

The composition may comprise any number of pharmaceutically acceptablespecies, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16of the listed species.

The concentration of the charged species is preferably within the rangeof from about 0.1% (w/w) to about 3.0% (w/w), such as from about 0.2%(w/w) to about 2.5% (w/w), from about 0.5% (w/w) to about 2.0% (w/w) orfrom about 1.0% (w/w) to about 1.5% (w/w).

Viscosity

The pharmaceutical composition of the invention is an aqueous gel. Thepolymer(s) within the composition form a network within which watermolecules are dispersed. The pharmaceutical composition of the inventionis preferably a hydrogel.

The aqueous gel has a viscosity in the range of from about 1000 to about500,000 micropascal-second (mPa·s) (also known as centipoises; cps) atroom temperature. Viscosity is a measure of the resistance of the gel tobeing deformed by either shear stress or tensile stress. Viscosity canbe measured using any method known in the art. Suitable methods include,but are not limited to, using a viscometer or a rheometer.

Room temperature is typically from about 18° C. to about 25° C., such asfrom about 19 ° C. to about 24° C. or from about 20° C. to about 23° C.or from about 21° C. to about 22° C. Room temperature is preferably anyof 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C. and 25° C.Viscosity is most preferably measured at 25° C.

The gel preferably has a viscosity in the range of from about 1000 toabout 500,000 mPa·s at room temperature, such as from about 1500 toabout 450,000 mPa·s at room temperature, from about 2000 to about400,000 mPa·s at room temperature, from about 2500 to about 350,000mPa·s at room temperature, from about 5000 to about 300,000 mPa·s atroom temperature, from about 10,000 to about 250,000 mPa·s at roomtemperature, from about 50,000 to about 200,000 mPa·s at roomtemperature or from about 50,000 to about 150,000 mPa·s at roomtemperature.

The gel most preferably has a viscosity in the range of from about50,000 to about 150,000 mPa·s (cps) at 25° C.

Preservatives

The pharmaceutical composition of the invention or the platelet lysateof the invention may further comprise one or more preservatives.Suitable preservatives are known in the art. Suitable preservativesinclude, but are not limited to, methylparaben, propylparaben andm-cresol.

Additional Growth Factors

In addition to the growth factors derived from the platelet lysate, thepharmaceutical composition of the invention or the platelet lysate ofthe invention may further comprise one or more additional or exogenousgrowth factors. Any growth factor may be present.

Additional amounts of one or more of, preferably all of, PDGF, VEGF,FGF, EGF, TGF, especially TGF-β, and CTGF may be added to thepharmaceutical composition of the invention or the platelet lysate ofthe invention.

The pharmaceutical composition of the invention or the platelet lysateof the invention may further comprise one or more antibiotics,analgesics or debriding agents. The pharmaceutical composition of theinvention or the platelet lysate of the invention may further comprisesilver

Cells

The pharmaceutical composition of the invention or platelet lysate ofthe invention may further comprise one or more stem cells. These canassist with the wound healing nature of the composition. The stem cellsmay be mesenchymal stem cells (MSC) or hematopoietic stem cells.

The pharmaceutical composition of the invention or platelet lysate ofthe invention may further comprise one or more progenitor cells,mononuclear cells or endothelial progenitor cells.

The pharmaceutical composition of the invention or platelet lysate ofthe invention may further comprise a progenitor cell of the mesodermallineage as described in International Application No. PCT/GB2012/051600.These cells express detectable levels of CD29, CD44, CD73, CD90, CD105and CD271 and do not express detectable levels of CD14, CD34 and CD45.

Any cells present in the composition or lysate are preferablyautologous. In other words, the cells are preferably derived from thepatient to which the cells will be administered. Alternatively, thecells are preferably allogeneic. In other words, the cells arepreferably derived from a patient that is immunologically compatiblewith the patient to which the cells will be administered.

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

“Platelet lysate” (aka “platelet released”) refers to the combination ofnatural growth factors contained in platelets that has been releasedthrough lysing those platelets.

“Protein,” “peptide,” and “polypeptide” are used interchangeably todenote an amino acid polymer or a set of two or more interacting orbound amino acid polymers.

“Stem cells” refers to any cell having the characteristic of beingunspecialized and able to renew for extended periods of time throughcell division and being inducible to become cells with specializedfunction.

“Lyophilisation” (aka freeze-drying, lyophilisation or cryodesiccation)is a dehydration process typically used to preserve a perishablematerial or make the material more convenient for transport.Freeze-drying works by freezing the material and then reducing thesurrounding pressure to allow the frozen water in the material tosublime directly from the solid phase to the gas phase. Methods forlyophilising compositions are known in the art.

Lyophilisation

Lyophilisation is advantageous in that the resulting substance is easierto store, can be kept in a small place, is easier to handle, can beapplied in a formulation (e.g., in a dry powder, an ointment, asuspension, a solution, a gel, a cream or a biocompatible, synthetic ornatural solid matrix) chosen according to the circumstances, can beadministered at an optimal dose, normally has a longer shelf life, andcan easily be screened for the most active preparation. One advantage oflyophilised cell extracts is that the material is readily availableexactly at the moment it is needed, in contrast to extract gels.

The invention also provides a platelet lysate produced using a method ofthe invention wherein the platelet lysate is lyophilised or in alyophilised form. The invention further relates to extracts of aplatelet lysate produced using a method of the invention wherein theplatelet lysate is lyophilised or in a lyophilised form. The methods ofproducing a platelet lysate of the invention may further compriselyophilising the platelet lysate. Methods suitable for lyophilising theplatelet lysate are known in the art.

Such extracts can be formed in a conventional manner. The term “extract”refers to a lysate product obtained from human cell lysate which hasretained its healing activity. Such extracts are discussed above. Aplatelet lysate extract can be prepared, for instance, after lysisand/or disruption of the blood cells followed by lyophilisation asdescribed above.

Such lyophilised compositions can be applied as a dry powder, in anointment, in a suspension, in a solution, in a gel, in a cream or in abiocompatible, synthetic or natural, solid matrix, chosen according tothe circumstances and administered in an optimal dose.

The pharmaceutical composition on the invention may be lyophilised or ina lyophilised form. The methods of producing a pharmaceuticalcomposition of the invention may further comprise lyophilising thepharmaceutical composition. Methods suitable for lyophilising thepharmaceutical composition are known in the art.

Medicaments, Methods and Therapeutic Use

A pharmaceutical composition of the invention or a platelet lysate ofthe invention may be used in a method of therapy of the human or animalbody. Thus, the invention provides a pharmaceutical composition of theinvention or a platelet lysate of the invention for use in treating awound, an anal fissure, vaginal atrophy or a wrinkle in a patient inneed thereof.

The invention also provides a method of treating a wound, an analfissure, vaginal atrophy or a wrinkle in a patient in need thereof,comprising administering to the patient a therapeutically effectiveamount of a pharmaceutical composition of the invention or a plateletlysate of the invention.

The invention concerns administering to the patient a therapeuticallyeffective amount of a pharmaceutical composition of the invention or aplatelet lysate of the invention. A therapeutically effective amount isan amount which ameliorates one or more symptoms of the wound, analfissure, vaginal atrophy or wrinkle. A therapeutically effective amountis preferably an amount which repairs the wound, anal fissure, vaginalatrophy or wrinkle. Suitable amounts are discussed in more detail below.

The pharmaceutical composition of the invention or the platelet lysateof the invention may be administered to any suitable patient. Thepatient may be any mammal. The patient is generally a human patient. Thepatient may be an infant, a juvenile or an adult. The patient istypically known to have wound, an anal fissure, vaginal atrophy or awrinkle.

The platelets used to prepare pharmaceutical composition of theinvention or the platelet lysate of the invention are preferablyautologous. In other words, the platelets are preferably derived fromthe patient to which the pharmaceutical composition of the invention orthe platelet lysate of the invention will be administered.Alternatively, the platelets are preferably allogeneic. In other words,the platelets are preferably derived from a patient that isimmunologically compatible with the patient to which the pharmaceuticalcomposition of the invention or a platelet lysate of the invention willbe administered.

The pharmaceutical composition of the invention or the platelet lysateof the invention used in therapy may comprise one or more stem cells orprogenitor cells of the mesodermal lineage as discussed above. It mayalso comprise additional growth factors as discussed above.

The invention may be used in combination with other means of, andsubstances for, treating the wound, anal fissure, vaginal atrophy orwrinkle or providing pain relief. In some cases, the pharmaceuticalcomposition of the invention or the platelet lysate of the invention maybe administered simultaneously, sequentially or separately with othersubstances which are intended for repairing the the wound, anal fissure,vaginal atrophy or wrinkle or for providing pain relief. Thepharmaceutical composition of the invention or a platelet lysate of theinvention may be used in combination with existing treatments for wound,anal fissures, vaginal atrophy or wrinkles and may, for example, besimply mixed with such treatments. Thus the invention may be used toincrease the efficacy of existing treatments or cosmetics.

Pharmaceutical Compositions and Administration

The pharmaceutical composition of the invention and platelet lysate ofthe invention may be formulated with pharmaceutically acceptablecarriers and/or excipients using routine methods in the pharmaceuticalart. The exact nature of a formulation will depend upon several factorsincluding the nature of the composition or lysate and the desired routeof administration. Suitable types of formulation are fully described inRemington's Pharmaceutical Sciences, 19th Edition, Mack PublishingCompany, Eastern Pennsylvania, USA.

The pharmaceutical composition of the invention or platelet lysate ofthe invention is typically sterile.

The pharmaceutical composition of the invention or the platelet lysateof the invention may be administered by any route. The pharmaceuticalcomposition of the invention or the platelet lysate of the invention istypically administered topically to the wound, anal fissure, vaginalatrophy or wrinkle.

The pharmaceutical composition of the invention or the platelet lysateof the invention may be prepared together with a physiologicallyacceptable carrier or diluent. Suitable carriers or excipients are, forexample, water, saline, dextrose, glycerol, of the like and combinationsthereof. Lyophilised compositions or lysates are typically rehydratedbefore therapeutic use.

In addition, if desired, the pharmaceutical composition of the inventionor the platelet lysate of the invention may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance effectiveness. Such agents areknown in the art.

The pharmaceutical composition of the invention or the platelet lysateof the invention are administered in a manner compatible with the dosageformulation and in such amount will be therapeutically effective. Thequantity to be administered depends on the subject to be treated,capacity of the subject's immune system and the degree repair desired.Precise amounts of required to be administered may depend on thejudgment of the practitioner and may be peculiar to each subject.

Any suitable amount of the pharmaceutical composition of the inventionor the platelet lysate of the invention may be administered to thesubject. For example, an amount sufficient to cover the wound, analfissure, vaginal atrophy or wrinkle is typically administered. Theactual amount administered will therefore depend on the size of thewound, anal fissure, vaginal atrophy or wrinkle. For instance, theamount of the pharmaceutical composition of the invention which isadministered may range from about 0.1 g to 100 g, such as from about 0.5g to about 75 g, from about 1 g to about 50 g, from about 2 g to about20 g or from about 3 g to 10 g.

The pharmaceutical composition of the invention or the platelet lysate,either of which may be in lyophilised form, may be therapeuticallyapplied to the wound. The processing of the platelets releases thegrowth factors into solution and the lyophilisation process wouldproduce a stabilized, freeze-dried pharmaceutical powder consisting ofthese factors. This lyophilised pharmaceutical composition or plateletlysate could be combined with other technologies for effective treatmentof clinical wound patients with much greater long term stability atdifferent temperatures. This would circumvent the logistical process ofpreparation and transfer of the treatment.

One embodiment is a dry, lyophilised pharmaceutical composition of theinvention or a dry, lyophilised platelet lysate. When combined withwater, just prior to treatment, a gel-like consistency would be formed.

In other embodiments, a pharmaceutical composition of the invention or aplatelet lysate of the invention could alternatively be combined withdifferent:

formulation/delivery methods, such as:

-   -   Lotion, Shake lotion, Cream, Ointment, Gel, Foam, Transdermal        patch, Powder, Solid, Sponge, Tape, Paste, bandage, gauze,        syringe, spray

treatments, such as:

-   -   Mesenchymal stem cells, hematopoietic stem cells, mononuclear        cells, endothelial progenitor cells, mesodermal progenitor        cells, antibiotics, analgesics, silver, debriding agents,        medical devices

Methods of packaging, such as:

-   -   Sterile package, bottle, box, can

Methods of storage

-   -   Ideally, room temperature powder; refrigerated or frozen, as        necessary

The invention still further relates to a pharmaceutical compositioncontaining, as an active substance, an extract as defined above, whichis preferably in lyophilised form and which can be used to promote thehealing of surface wounds, for example of skin, such as human skin. Thispharmaceutical composition preferably comprises the lyophilised extractin a formulation suitable for application onto surface wounds. Such aformulation can be applied directly to a surface wound either as a drypowder or in the form of a gel, a cream, an ointment, a suspension, asolution, or a biocompatible, synthetic or natural solid matrix, any ofwhich can be prepared in a conventional manner, if desired withconventional, pharmaceutically acceptable excipients and additives.Normally, the lyophilised extract is incorporated in such a compositionin a concentration that depends on of the type of surface wound to behealed and the circumstances, under which the composition is to be used.For instance, a lyophilised platelet lysate extract of this inventioncan be applied at a concentration, such that the amount of activesubstance for wound healing, per cm², is equivalent to the amount ofactive substance found in the preparation.

Wounds

The wound to be treated in accordance with the invention is preferably askin wound. The skin wound is preferably an ulcer.

Examples of types of wounds which can be treated with the pharmaceuticalcomposition or platelet lysate of this invention include, but are notlimited to, thermal, chemical, electrical and radiation-induced burnwounds of skin; burn wounds covered with meshed skin autografts, fullthickness and partial thickness, mechanical wounds such as incisions,abrasions and lacerations; these types of wounds include also surgicaland excision wounds in skin; various ulcerations of skin, such asdecubitus, venous and arterial ulcers and ulcers caused by underlyingdiseases such as diabetes and vasculitis; corneal wounds; tympanicmembrane lesions; and lesions due to pathological conditions such asbullous pemphigoid, epidermolysis bullosa and lupus erythomatosus.

This invention yet further relates promoting the healing of a surfacewound (for example in skin, e.g., human skin) by applying thepharmaceutical composition of this invention to the surface of thewound.

Anal Fissure

An anal fissure is a break or tear in the skin of the anal canal. Such afissure may be treated by applying a pharmaceutical composition of theinvention or a platelet lysate of the invention to the fissure.

Vaginal Atrophy

Atrophic vaginitis (also known as vaginal atrophy or urogenital atrophy)is an inflammation of the vagina (and the outer urinary tract) due tothe thinning and shrinking of the tissues, as well as decreasedlubrication. It is typically caused by a decrease in secreation of thehormone estrogen. The atrophy may be treated by topically applying apharmaceutical composition of the invention or a platelet lysate of theinvention.

Wrinkle

Wrinkles treated in accordance with the invention are typically skinwrinkles. A wrinkle may be treated by applying a pharmaceuticalcomposition of the invention or a platelet lysate of the invention tothe wrinkle.

The following Examples illustrate the invention.

EXAMPLE 1 Platelet Lysate

A sample of whole blood was collected and saved for analysis of thenumber of platelets, as a way to monitor the lysis process. The wholeblood was centrifuged (120×g, 15 minutes, no break, room temperature) toseparate the platelets from the remaining blood cells. The plateletsended up in the yellow plasma, known as platelet rich plasma (PRP),which was resting on top of a dark red pillar of the remaining bloodcells.

A small sample of the PRP was saved to analyze the platelets, and it wasshow that the platelet concentration is higher compared to the wholeblood as the platelets have now been concentrated in a smaller volume.The volume of the plasma differs between individuals but is related togender such that the volume will be smaller in men and the plateletconcentration in whole blood compared to PRP will therefore haveincreased more in men.

The PRP was transferred to another container, such as a 50 ml tube, andwas then submerged in liquid nitrogen (−196° C.) until frozen or for 5minutes. The tube was thereafter transferred to a 37° C. water bath, orother source of heat such as an incubator, until the PRP had thawed.This was referred to as one liquid nitrogen freeze/thaw cycle. Thiscycle was repeated three (3) more times for a total of four (4)freeze/thaw cycles. After these cycles the resulting product wasreferred to as C4 PL (platelet lysate) produced through 4 freeze/thawcycles, and upon analysis it contained about 5% of the starting numberof platelets in the PRP. The C4 PL contained gel lumps of plateletdebris which can be removed by transferring the C4 PL to a new tube (theheavy gel lumps will fall to the bottom of the tube) or centrifuging it(3200×g, 20 minutes, with break) and using the supernatant. The C4 PLalso contained microscopic debris which was visible under a microscope.

EXAMPLE 2 Platelet Lysate Gel

Platelet lysate prepared as in Example 1 was transferred into agraduated sterile Falcon tube. If previously frozen, the platelet lysatewas first thawed at room temperature. To the platelet lysate, calciumgluconate and plasma or thrombin were added in appropriate proportions.If the plasma was previously frozen, it was thawed at 37° C. prior touse. Appropriate proportions as were used herein include: e.g. 1:5 partsof platelet lysate, 2 parts of plasma, 2 parts of calcium gluconate; ande.g. 2:3 parts of platelet lysate, 1 part of thrombin, and 0.5 parts ofcalcium gluconate. The resulting suspension was exposed to careful slowshaking to complete 10-12× 360° tube revolutions and then fractionatedby size into sterile dispensation devices. Devices included syringes andmicro-needle dispensers. In some instances fractionation by size wasdetermined according to the size and shape of the ulcer to be treated.

EXAMPLE 3 Platelet Lysate Gel Utility

For treatment of vaginal atrophy: 2-10 ml of plasma lysate gel withviscosity of ˜1000 cps was applied intra-vaginally daily or every otherday for 1-2 weeks. This was repeated for a week if symptoms recurred.

For treatment of a 5 mm diameter Diabetic foot ulcer: 2-3 ml of plasmalysate gel with viscosity of 70,000-100,000 cps was applied topicallyonto the ulcer daily every other day for 2-3 weeks.

REFERENCES

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1-11. (canceled)
 12. A method of producing a platelet lysate comprisingsubjecting a population of platelets to at least one freeze-thaw cycle,wherein the freeze portion of each cycle is carried out at a temperaturelower than or equal to −78° C.
 13. A method according to claim 12,wherein the method comprises subjecting the population of platelets tothree or four freeze-thaw cycles.
 14. A method according to claim 12,wherein liquid nitrogen is used as a cryogenic means whereby immersionin the freeze portion of each cycle results in 95% or more lysis ofplatelets resulting in greater growth factor release and improvedfunction in cell growth, repair and regeneration measurable by but notlimited to fibroblast and PGDF assay.
 15. A method according to claim12, where dry ice (solid carbon dioxide) and an organic solvent are usedas a cryogenic means in the freeze portion of each cycle.
 16. A methodof producing platelet lysate comprising subjecting a population ofplatelets to mechanical homogenisation (‘waring blender’), liquidhomogenization, sonication, freeze-thaw using alcohol and dry ice orother means, mechanical grinding in liquid nitrogen at >75%, buttypically >90%, lysis to release growth factors from platelets and alphagranules into solution whereas rotating blades grind and disperse cellsand tissues; cell or tissue suspensions are sheared by forcing themthrough a narrow space; high frequency sound waves shear cells; repeatedcycles of freezing and thawing disrupt cells through ice crystalformation; grinding tissue, frozen in liquid nitrogen wherein lysisreleases growth factors from platelets and alpha granules into solution.17. A method according to claim 12 or 16, wherein the method furthercomprises lyophilising the platelet lysate.
 18. A platelet lysateproduced using a method according to claim 12 or
 16. 19. A plateletlysate according to claim 18 for use in treating a wound, an analfissure, vaginal atrophy or a wrinkle in a patient in need thereof. 20.A method of treating a wound, an anal fissure, vaginal atrophy or awrinkle in a patient in need thereof, comprising administering to thepatient a therapeutically effective amount of a platelet lysateaccording to claim
 18. 21-22. (canceled)